| BACGROUND: Atherosclerosis is an artery disease related to multiple factors and abnormalities of many genes, which is charactered by disorder of cholesterol transport and metabolism. The formation of foam cells is the core event of atherosclerosis development. The disorder of cholesterol metabolism is involved in the whole course of the foam cell formation. The scavenger receptor class B type I (SR-BI),which is a kind of glycoproteins on the cell membrane, plays very important role in the development of atherosclerosis. SR-BI expressed in liver promotes the selective uptake of high density lipoprotein-cholesteryl ester (HDL-CE), an important route of the reverse cholesterol transport (RCT). High density lipoprotein (HDL) has been reported showing antiatherogenic effect through many mechanisms. It was previously shown that phospholipids-enriched HDL facilitates cellular cholesterol efflux, reduces cholesterol accumulation. And the results show that the physical state of the PL molecules in the rHDL particles significantly affects the ability of the particles to remove cell FC. The study is aimed to investigate the effect of lipoprotein which was reconstituted by different proportion phospholipid on cholesterol transport and the expression of SR-BI.OBJECTIVE To investigate the effects of rHDL-A1,rHDL-A2,rHDL-A3,rHDL-B1,rHDL-B2,HDL3 on cholesterol traffic in J774 macrophages, the effects of rHDL-A1,rHDL-A2,rHDL-A3 on the expression of SR-BI in J774 macrophages, the effects of rHDL-A3 with different concentration on the expression of SR-BI in J774 macrophages.METHODS Low density lipoprotein (1.006-1.063g/ml), high density lipoprotein 2 (1.063-1.125g/ml), and high density lipoprotein 3 (1.125-1.21g/ml) were prepared by ultracentrifugation. Oxidized modification of LDL was carried out through incubation with 10μmol/L CuSO4 for 24h, followed by PBS dialyzing three times at 1:500 volume ratio. The reconstituted lipoprotein with different composition content was prepared by mixed compound of POPC,CE,Apo A-I and sodium cholate at optimaized conditions of reaction.The experiments were designed as follows: The first, investigate the effects of rHDL-A1,rHDL-A2,rHDL-A3,rHDL-B1,rHDL-B2 and HDL3 on cholesterol traffic in J774 macrophages. Cells which preincubated with 60μg/ml OxLDL for 24hr were incubated with rHDL-A1,rHDL-A2,rHDL-A3,rHDL-B1,rHDL-B2 and HDL3 for 16hr. Cellular cholesterol transport was determined high performance liquid chromatography analysis, [3H]-labeled Cholesterol efflux was determined by FJ-2107P type liquid scintillator.Second, study the effect of rHDL-A1,rHDL-A2,rHDL-A3 on expression of SR-BI in J774 macrophages,which was analyzed through reverse transcription polymerase chain reaction (for mRNA expression) and Western blotting (for protein expression).Third, Detect the effect of rHDL-A3 of different concentration on expression of SR-BI in J774 macrophages, which was analyzed through reverse transcription polymerase chain reaction (for mRNA expression) and Western blotting (for protein expression).RESULTS High performance liquid chromatography analysis demonstrated the ratios of cellular CE/TC were 49%,23%,39%,36%,32%,29%,24%(P<0.05), the results showed that the ratios of cellular CE/TC were reduced with increase of the phospholipids in reconstituted lipoprotein (J774 macrophages which were pre-induced by oxLDL incubated with reconstituted lipoprotein contained different proportion phospholipids). Compared with the control group which were pre-incubated by oxLDL, the ratios of cellular CE/TC of HDL3 group was down. The percentage of each group cholesterol efflux were 5.24%,26.59%,15.47%,18.66%,22.57%,20.36%,11.46%(P<0.05). We can see from the results that the percentages of cholesterol efflux were up gradually by increase of phospholipids in reconstituted lipoprotein. When the phospholipids in lipoprotein was increased, the expression of SR-BI in J774 macrophages which incubated with rHDL-A1,rHDL-A2,rHDL-A3 was up-regulated by concentration-dependent manner gradually.SUMMARY With the increasing of the content of phospholipid composition in reconstituted lipoproteins, the ratios of cellular cholesteryl ester to total cholesterol (CE/TC) were reduced. HDL3 decreased the ratio of cellular CE/TC compared with the control group which was pre-incubated by oxLDL. In present experiments reconstituted lipproteins (rHDL-A1,rHDL-A2,rHDL-A3,rHDL-B1,rHDL-B2) decreased cellular cholesterol accumulation in J774 macrophages. However, HDL3 group showed the most effect of cholesterol accumulation reduction. With increasing of the phospholipid proportion in reconstituted lipoproteins, the percentage of cholesterol efflux was increased. The experiment also showed that the increase of phospholipid content in the lipoproteins, the expression of SR-BI in J774 macrophages which incubated with rHDL-A1,rHDL-A2,rHDL-A3 was up-regulated with concentration-dependent manner.CONCLUSIONS In this paper we have demonstrated that with the increasing of the content of phospholipid composition in reconstituted lipoproteins, the cholesterol efflux of J774 macrophages is increased. Increase of the content of cholesteryl ester composition in reconstituted lipoproteins which contain the same proportion of phospholipids, the cellular cholesterol efflux of J774 macrophages is reduced. The expression of scavenger receptor class B type I (SR-BI) in macrphages was up-regulated by phospholipid.
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